A multiplexed microflow LC–MS/MS-PRM assay for serologic quantification of IgG N- and HPX O- glycoforms in liver fibrosis

Targeted quantification of glycoproteins has not reached its full potential because of limitations of the existing analytical workflows. In this study, we introduce a targeted microflow LC–MS/MS-PRM method for the quantification of multiple glycopeptides in unfractionated serum samples. The entire preparation of 16 samples in a batch is completed within 3 h, and the LC–MS quantification of all the glycoforms in a sample is completed in 15 min in triplicate, including online capture and desalting. We demonstrate applicability of the workflow on a multiplexed quantification of eight N-glycoforms of immunoglobulin G (IgG) together with two O-glycoforms of hemopexin (HPX). We applied the assay to a serologic study of fibrotic liver disease in patients of HCV etiology. The results document that specific IgG- and HPX-glycoforms detect efficiently fibrotic disease of different degree, and suggest that the LC–MS/MS-PRM assays may provide rapid and reproducible biomarker assay targeting simultaneously the N- and O-glycoforms of the peptides. We propose that such high throughput multiplexed methods may advance the clinical use of the LC–MS/MS assays.

www.nature.com/scientificreports/ optimized method allows complete processing (reduction, alkylation and tryptic digestion of proteins) of the unfractionated serum samples in approximately 3 h using Pressure Cycling Technology (Barocycler NEP2320 EXT, Pressure BioSciences, South Easton, MA) 19 which is followed by a 5 min analysis of each sample by a targeted LC-MS/MS-PRM assay with online analyte capture, desalting, and gradient elution. We used the method to quantify selected IgG N-glycoforms and HPX O-glycoforms in serum samples of patients with HCV-induced fibrotic liver disease, using a capillary flow LC system (Dionex Ultimate 3000) and Q Exactive-HF mass spectrometer (Thermo). We demonstrate utility of the method in a high-throughput serologic screening setup which opens up the potential for clinically relevant serologic screening of glycopeptide biomarker candidates in the fibrotic liver disease.

Experimental section
Materials and reagents. Ammonium  Sample processing. Serum samples from control, fibrosis and cirrhosis groups were processed directly by trypsin digestion, without any enrichment step 20 . Briefly, serum samples (2 µl each) were diluted 1:70 with 25 mM ammonium bi-carbonate; and treated with 5 mM DTT at 60 O C for 1 h, followed by 15 mM iodoacetamide for 20 min at RT in the dark, then 5 mM DTT for 20 min at RT. Proteins in a fixed volume of samples from above (20 µL of the reduced and alkylated diluted serum sample) were digested with mass spectrometry grade Trypsin/Lys-C mix (1 µg) at 37 °C in a Barocycler (60 cycles, 1 min hold at 30kpsi). The peptides were analyzed by LC-MS/MS without any additional processing steps.

Micro-flow LC-MS/MS-PRM.
LC-MS/MS analysis was performed using capillary-flow Ultimate 3000 RSLCnano chromatography system and a Q-Exactive HF Mass Spectrometer (Thermo) with a nanospray source housing a multinozzle M3 emitter spray tip (Newomics, Berkeley, CA, USA) 21 . The samples were loaded onto a PepMap C18 Cartridge (1 mm x 5 mm) and desalted using a sample loading pump at 10 μl/min flow rate 0-1 min with 0.1% formic acid in water, following which the peptides were eluted at a 5 μl/min flowrate. A C18 Acclaim PepMap 100 75 μm × 2 cm nanoViper column was directly connected to the multinozzle emitter. A schematic of the gradient ( Supplementary Fig. S1) consists of a 1-2.5 min linear gradient of 1-15% ACN in 0.1% aqueous formic acid, followed by 30 s to 90% ACN, then hold at 90% ACN for 1 min, and equilibration of the column for 1 min at 0% ACN. The valve is switched at the equilibration step, and the trap column is equilibrated at 10 μl/min flow rate making it ready for the next sample injection. A Parallel Reaction Monitoring (PRM) workflow was used for scheduled MS/MS fragmentation of target ions (isolation window m/z 2.0, HCD fragmentation, resolution 30 K). Table 1 shows the peptide glycoforms analyzed in this study, MS data collection parameters, and transitions used for the quantitation. We analyzed simultaneously selected IgG N-glycoforms of the peptide EEQYNSTYR (G0, G0N, G0FN, G0F, G1, G1N, G1FN, G1F), and mono-and di-sialyated HPX O-glycopeptide TPLPPTSAHGNVAEGETKPDPVTER HexNAc(1) Hex(1)Neu5Ac(1), HexNAc(1)Hex(1)Neu5Ac(2) 11,20,21 . The respective glycan structures are shown in Table 1.
Study population. The sample set was described in our previous study 22 . Briefly, serum samples of participants in the HALT-C trial, whose fibrotic status was determined by ISHAK score based on liver biopsy, were obtained from the central repository at the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK). The HALT-C trial is a prospective randomized controlled trial of 1050 patients that evaluated the effect of long-term low-dose peginterferon alpha-2a in patients who failed initial anti-HCV therapy with interferon 23 . All the patients in our study were from the control arm of the trial and were compared to diseasefree controls frequency matched on age, race and gender; that the controls donated blood samples at Georgetown University (GU) in line with an approved IRB protocol. In this study, we analyzed 15 disease free controls, 15 HCV fibrotic (Ishak score 3-4), and 15 HCV cirrhotic (ISHAK score 5-6) patients. (Supplementary Table S1). Data analysis. LC-MS/MS data were processed by Quan Browser (Thermo) to deduce the peak areas based on the listed transition ions ( Table 1). The peak areas of the IgG glycoforms were normalized against the log 2 peak area of an internal IgG peptide GPSVFPLAPSSK, quantified simultaneously. The internal peptide peak area was calculated by summing the intensity of three product ions. For the measure of O-HPX the ratio of disialoT-(HexNAc(1)Hex(1)Neu5Ac(2) over monosialoT-HexNAc(1)Hex(1)Neu5Ac(1) was calculated based on their respective peak areas and defined as S-HPX, as described previously 20 .
Statistical analysis of the datasets was performed using GraphPad Prism software (v9.4.1). The distribution of normalized peak areas of the IgG glycoforms, and S-HPX were compared between the disease groups, and the data was visualized by a nested Tukey plot. The descriptive statistics such as minimum, maximum, mean, and standard deviation were summarized for each analyte. One-way ANOVA tests, two-sample t-tests, and the area under the receiver operating characteristic curve (AuROC) along with sensitivity and specificity were used to evaluate the ability of each analyte to separate the disease groups. In parallel, Kruskal-Wallis and Wilcoxon tests were used to account for non-normality in analytes. A two-sided significance level of 0.05 was used for statistical significance.

Results and discussion
In this study, a multiplexed microflow LC-MS/MS PRM method was developed to quantify simultaneously selected IgG N-glycopeptides and sialylated HPX O-glycopeptides in a 5 min run at a 5 µl/min flow rate. We do this because the fibrotic liver pathology leads to changes in the secreted proteome and its glycosylation which can be efficiently captured by serologic analysis of the glycopeptides. Our prior studies showed that both N-glycosylation and O-glycosylation pathways are altered 20,22,24,25 and we expected that their simultaneous quantification will provide an improved reflection of the liver pathology. However, fast and robust analytical methods is a prerequisite for a clinically relevant assay which prompted us to optimize this multiplexed fast method.
A standard QE-HF mass spectrometer coupled to Dionex capillary flow LC system proved adequate as the analytical equipment of choice, and the introduction of the analytes to the MS was improved by the use of a multinozzle M3 emitter spray tip (Newomics) 21 . The higher flow rate in mFlow-LC reduces the gradient time in comparison to traditional nanoflow LC-MS/MS, and increases the reproducibility and robustness of the measurements 26,27 . Use of a multinozzle emitter tip that splits the flow evenly into multiple smaller streams enhance the ionization efficiency 28 . This helped us develop a fast, robust method for a multiplex analysis of Nand O-glycopeptides in one analytical run using commonly available equipment. This means that this type of assay can now be adapted for clinical-type sample screening in other laboratories which is one of the goals of our www.nature.com/scientificreports/ study. Since the IgG and HPX proteins are abundant in serum, the readily available QE-HF mass spectrometer is fully adequate compared to more sensitive mass analyzers on the market or used in our previous studies 11,20,21,25 . To achieve time efficiency, sample preparation from crude serum was performed without any pre-fractionation steps using pressure cycling in a barocycler. This enabled us to complete the sample preparation and MS sample analysis on the same day. The throughput could be further improved by implementing fast reduction, alkylation and sample digestion in one-step using accelerated barocycler techniques 29,30 . LC-MS analysis was performed using an efficient 5 min microflow gradient. Online analyte captures and desalting was achieved using a trap cartridge at 10 µl min flow rate, followed by analysis using a column connected directly to a multinozzle spray emitter. Sample elution was performed with a 2 min gradient, followed by column regeneration for 1 min, and equilibration for 1 min which is used simultaneously for the next sample loading. This allowed us to complete a sample-run in triplicate in 15 min. This is a highly efficient setup especially in view of the fact that we multiuplex quantification of relevant N-and O-glycopeptides in the same run. Quantification of sialylated O-glycoforms of HPX was performed as it is a potential biomarker; we did not observe changes in sialylated N-glycoforms of IgG in the context of fibrotic liver disease, thus it was not a target in current analysis.
We targeted selected glycoforms of IgG N-glycopetide EEQYN 297 STYR, namely G0-HexNAc(4)Hex (3) Table 1). The product ions for the IgG N-glycopeptides were collected at low energy HCD setting 11 . The peak areas of G0, G0N, G0F, and G0FN were calculated from the product ions which resulted from a selective loss of one HexNAc from the parent glycopeptide; the G1, G1F, G1FN, and G1N products resulted from the loss of one HexNAc-Hex fragment. The peak areas of the O-HPX glycoforms were calculated from the loss of the glycan resulting in a peptide backbone transition 20 . The elution profiles of selected informative analytes are shown in Fig. 1.
As a proof of principle, we used the assay to quantify the analytes in the serum of fibrotic (n = 15) and cirrhotic (n = 15) hepatitis C patients compared to a group of disease-free controls (n = 15). The peak areas of the IgG N-glycoforms were normalized to the average peak area of an internal IgG peptide. S-HPX was used as the measure of the O-HPX glycoforms, as described in the methods 20,21 . Statistical analyses were performed to assess the association between the analytes and disease groups and the mean, standard deviation and p-values from one-way ANOVA analysis is shown in Table 2. The observed p-values for the analytes G0F, G0FN, G1FN, and S-HPX were < 0.001.
T-test was performed to assess the association of each analytes between control vs fibrosis, fibrosis vs cirrhosis, and control vs cirrhosis groups (Supplementary Results- Table S2). All of the analytes measured in this study separated control vs cirrhosis group (p ≤ 0.004, except G1N p = 0.017). Most IgG analytes did not separate efficiently fibrotic and cirrhotic patients (p > 0.05), except G1 (p = 0.02) and G1N (0.05). But they (except G1 and G1N) separate the controls from the fibrotic group. The G0FN (p < 0.001) and G0F (p < 0.001) glycoforms separate these two groups most efficiently as shown by a nested tukey plot (Fig. 3); please see supplementary figure S2 for the other IgG analytes. To the contrary, the S-HPX separates efficiently the fibrosis and cirrhosis groups (p = 0.01) but is less efficient in separating the control and fibrosis groups groups (Fig. 4).
The results from above statistical analysis indicate that a combination of the IgG G0FN and G0F with S-HPX may provide an efficient way to assess progression of the fibrotic liver disease from mild to advanced stages. The independent AuROC analyses using the two IgG N-glycoforms to compare normal vs. fibrotic groups and S-HPX to compare the fibrotic vs. cirrhotic groups confirms that. The G0FN and G0F glycoforms achieve AuROCs of 0.92 and 0.87, respectively, in separating the fibrosis from control groups and the S-HPX separated cirrhosis from control with AuROC of 0.84 ( Fig. 5 and Supplementary Table S3).
To summarize, we optimized multiplex analysis of the N-glycoforms of IgG and O-glycoforms of HPX in one analytical run. The simultaneous analysis of the two classes of glycosylated peptides is informative for serologic assessment of liver fibrosis and is completed in 5 min which further reduces time of analysis by 8 min compared to our previous report 21 , a 2.5-fold improvement in time efficiency. We also transferred the analyses to a common LC-MS/MS platform which can be adopted for routine testing. This study measured the N-and O-glycoforms in one LC-MS run which allowed an efficient serologic quantitative assessment of the advancing liver fibrosis by assessment of the two independent glycosylation pathways simultaneously. We propose that the use of the IgG G0FN and G0F in combination with S-HPX can be readily evaluated as a means of non-invasive serologic assessment of liver fibrosis in large clinically relevant datasets. We anticipate that the microflow LC-MS/MS-PRM methods will be a useful avenue for the quantification of various glycopeptides in biological and clinical materials.